The human skin fibroblast appears to be an androgen-responsive system. Thus this cell has the enzyme 5 alpha-reductase and an androgen receptor (AR) system; and our preliminary results suggest androgen-mediated stimulation of cell multiplication. The purpose of this project is to characterize the androgen receptor system in those cells. Subcellular localization of the 5 alpha-dihydrotestosterone-AR complex (DHT-AR) will be determined by fractionation of cell homogenates and nuclear preparations obtained from fibroblasts exposed to 3H-DHT. The DHT-AR complex formed in cell sonicates at 4 degrees appes to differ from that formed at 37 degrees in the intact cell. This change in DHT-AR results in the binding of the complex, apparently to chromatin, and an increased affinity of the complex for hydroxylapatite. These findings will be further documented by subcellular localization. Also the nature of the change in the complex (activation) will be defined by measurement of various physical-chemical parameters of the two species of DHT-AR complex. Among the parameters to be measured are sedimentation velocity, isoelectric pH, and mobility on gel filtration and ion exchange columns. Partial purification of the native AR and activated AR' will be carried out to enable measurement of the parameters of the kinetics, thermodynamics and stereochemistry of the androgen binding reaction, as well as the physico-chemical properties of the DHT-AR and DHT-AR' complexes. The effects of a variety of antiandrogens on the androgen binding reaction, and on the stability, the activation and the chromatin binding of the complex will be determined. It is believed that these studies will lead to the development of a model system in which to study: 1) the regulation of androgen-mediated events, 2) the analysis of the causes of male pseudohermaphroditism and hypospadias and 3) the development of the pharmacology of antiandrogens and partial androgen agonists. The last could lead to improvements in the therapy of virilizing syndromes and in the prediction of teratogenic potential of synthetic steroids.